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Directed evolution of aminoglycoside phosphotransferase (3') type IIIa variants that inactivate amikacin but impose significant fitness costs.

ABSTRACT: The rules that govern adaptive protein evolution remain incompletely understood. Aminoglycoside aminotransferase (3') type IIIa (hereafter abbreviated APH(3')-IIIa) is a good model enzyme because it inactivates kanamycin efficiently; it recognizes other aminoglycoside antibiotics, including amikacin, but not nearly as well. Here we direct the evolution of APH(3')-IIIa variants with increased activity against amikacin. After four rounds of random mutation and selection in Escherichia coli, the minimum inhibitory concentration of amikacin rose from 18 micrograms/mL (wild-type enzyme) to over 1200 micrograms/mL (clone 4.1). The artificially evolved 4.1 APH(3')-IIIa variant exhibited 19-fold greater catalytic efficiency (k cat/K M) than did the wild-type enzyme in reactions with amikacin. E. coli expressing the evolved 4.1 APH(3')-IIIa also exhibited a four-fold decrease in fitness (as measured by counting colony forming units in liquid cultures with the same optical density) compared with isogenic cells expressing the wild-type protein under non-selective conditions. We speculate that these fitness costs, in combination with the prevalence of other amikacin-modifying enzymes, hinder the evolution of APH(3')-IIIa in clinical settings.

SUBMITTER: Kramer JR

PROVIDER: S-EPMC3800079 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Directed evolution of aminoglycoside phosphotransferase (3') type IIIa variants that inactivate amikacin but impose significant fitness costs.

Kramer Joseph R JR Matsumura Ichiro I

PloS one 20131018 10

The rules that govern adaptive protein evolution remain incompletely understood. Aminoglycoside aminotransferase (3') type IIIa (hereafter abbreviated APH(3')-IIIa) is a good model enzyme because it inactivates kanamycin efficiently; it recognizes other aminoglycoside antibiotics, including amikacin, but not nearly as well. Here we direct the evolution of APH(3')-IIIa variants with increased activity against amikacin. After four rounds of random mutation and selection in Escherichia coli, the mi ...[more]

PMID: 24204655

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Project description:BackgroundDespite increasing documentation of insecticide resistance in malaria vectors against public health insecticides in sub-Saharan Africa, there is a paucity of information on the potential fitness costs of pyrethroid resistance in malaria vectors, which is important in improving the current resistant management strategies. This study aimed to assess the fitness cost effects of insecticide resistance on the development and survival of immature Anopheles gambiae from western Kenya.MethodsTwo-hour old, first instar larvae (L1) were introduced and raised in basins containing soil and rainwater in a semi-field set-up. Each day the number of surviving individuals per larval stage was counted and their stage of development were recorded until they emerged as adults. The larval life-history trait parameters measured include mean larval development time, daily survival and pupal emergence. Pyrethroid-resistant colony of An. gambiae sensu stricto and susceptible colony originating from the same site and with the same genetic background were used. Kisumu laboratory susceptible colony was used as a reference.ResultsThe resistant colony had a significantly longer larval development time through the developmental stages than the susceptible colony. The resistant colony took an average of 2 days longer to develop from first instar (L1) to fourth instar (L4) (8.8 ± 0.2 days) compared to the susceptible colony (6.6 ± 0.2 days). The development time from first instar to pupa formation was significantly longer by 3 days in the resistant colony (10.28 ± 0.3 days) than in susceptible colony (7.5 ± 0.2 days). The time from egg hatching to adult emergence was significantly longer for the resistant colony (12.1 ± 0.3 days) than the susceptible colony (9.6 ± 0.2 days). The pupation rate (80%; 95% (CI: 77.5-83.6) vs 83.5%; 95% (CI: 80.6-86.3)) and adult emergence rate (86.3% vs 92.8%) did not differ between the resistant and susceptible colonies, respectively. The sex ratio of the females to males for the resistant (1:1.2) and susceptible colonies (1:1.07) was significantly different.ConclusionThe study showed that pyrethroid resistance in An. gambiae had a fitness cost on their pre-imaginal development time and survival. Insecticide resistance delayed the development and reduced the survivorship of An. gambiae larvae. The study findings are important in understanding the fitness cost of insecticide resistance vectors that could contribute to shaping resistant management strategies.

Dataset Information

Directed evolution of aminoglycoside phosphotransferase (3') type IIIa variants that inactivate amikacin but impose significant fitness costs.

Publications

Directed evolution of aminoglycoside phosphotransferase (3') type IIIa variants that inactivate amikacin but impose significant fitness costs.

Similar Datasets

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